Just add water: Rainfall‐induced anther closure and color change in Ripariosida hermaphrodita (Malvaceae)

Abstract Anther opening has commonly been thought of as unidirectional, but reports of anthers closing in response to rainfall show this is not the case. In some species, anther closure can protect pollen from degrading or washing away, thus possibly enhancing male fitness. Similarly, although floral color is often presumed to be static, numerous floral parts may change color during blooming. These color changes primarily occur in response to pollination or aging, thus potentially increasing pollination efficiency by directing floral visitors to recently opened, unpollinated flowers. Daily observations of 364 Ripariosida hermaphrodita flowers from seven individuals showed that anthers that were purple, open, and shedding pollen became beige colored and tightly closed after rainfall. These findings were further supported by observations of plants exposed to simulated rainfall in a greenhouse and time‐lapse photography of flowers misted with water. To our knowledge, our work represents the first report of anther closure in response to rain in Malvaceae and the first report of floral color change induced by rainfall.

biology and reproduction.
Here, we report novel observations of rainfall-induced anther closure and color change in R. hermaphrodita. Anther closure in response to rain has not previously been reported in Malvaceae. To our knowledge, rainfall-induced floral color change has never before been observed. Together, these findings begin to characterize this rare plant's reproductive biology and provide new insight into how dynamic abiotic factors may affect floral evolution. 2 | ME THODS

| Floral observations
We present data from seven R. hermaphrodita plants that were observed from July 11 to September 8, 2022. The plants were grown from seed, potted up, and maintained outdoors, with regular watering. The seed originated from three wild-collected populations and one cultivated source of unknown provenance. Flowers were ob- To be included in our analyses, floral data had to be "complete," meaning they met the following criteria: at least one observation was made of the flower before the petals had expanded enough to reveal the androecium, daily observations were recorded while the flower was in bloom, and a final observation indicated the flower had senesced, here defined as the abscission of the basally fused unit of the corolla + androecium. Data were rendered incomplete for many reasons, including the loss of a label, lateral shoot damage, floral herbivory, and missed days of observations.

| Natural rainfall data
Rainfall records were obtained through National Weather Service's NOWData (National Weather Service, 2022). Data for "Philadelphia Area, PA" were downloaded on October 12, 2022. These data were recorded at Philadelphia International Airport which is roughly 16 km from the study site. For days where precipitation was present but quantities were too small to measure, these records reported "Trace" rainfall. We treated "Trace" rainfall as 0 cm. We define a "rain event" as a date or continuous string of dates with greater than "Trace" rainfall. Initially, direct observations of rain events at the study site were not recorded because we were not anticipating R. hermaphrodita would display rainfall responses when the observation protocol was designed.

| Simulated rainfall experiment
We conducted a simulated rainfall experiment on September 13, 2022. Six plants of R. hermaphrodita were housed in a greenhouse and watered only at the soil level to ensure the flowers in anthesis had not been exposed to water. On each plant, the lowest six flowers that met experimental criteria were observed. These criteria required flowers to be in prime bloom, have pollen on their anthers, be free from damage, and be low enough on the plant that water falling from our overhead watering system would reach them. One plant had only three flowers that met these criteria, so only these three were included in experimentation. The six plants were made up of three pairs, with each pair representing plants grown from seed from the same population. One plant from each pair was chosen at random to be in the experimental group with the other being in the control group. For each flower, anther color and position were recorded.
Then the three plants selected for experimental treatment were watered for 30 min with an overhead watering system. Immediately

| Data analysis
All observations of anther position in R. hermaphrodita flowers were as- Similarly, observations of anther color were assigned to three categories: "purple" if most anthers were purple; "beige" if most anthers were translucent, white, beige, yellow, or brown; or "mixed" if there was a nearly even mix of purple and beige anthers, the anthers were an intermediate purple-beige, or anther color could not be determined. This Chi-squared test with Yate's continuity correction was used to test for statistically-significant differences in anther color and position change in the flowers of the experimental group compared to those of the control group. So few anthers showed greater pigmentation or a more-closed anther position that these data would have rendered these Chi-squared tests unreliable; therefore, they were omitted in both the experimental and control group. A second Chisquared test was used to compare anthers before and after treatments for the simulated rainfall data. For the natural rainfall data, standard error was calculated for average cumulative rainfall totals and used to compare rain events associated and not associated with transitions in anther color and position across >88% of flowers.

| Time-lapse photography
Multiple series of photographs were taken of the anthers of R. hermaphrodita flowers and combined into time-lapse videos. These flowers were sprayed with water to simulate a rain event. Photographs were taken with a Canon EOS 5DS camera connected to a Zeiss Stemi 2000-C microscope at a magnification that allowed anthers to fill, but not exceed, the frame (approximately 25× magnification).
Flowers were clipped from potted plants and brought to the microscope area. To improve visibility, sufficient anthers were trimmed from flowers to leave a (roughly) single horizontal plane of anthers. If an abundance of pollen obscured the view of the remaining anthers, some pollen was removed to reveal more of the anther walls.
Variables such as time-lapse length, number of photos, magnification, video angle, and frequency of water application were manipulated to determine what combination best showed the phenomenon of interest. Ultimately, two time-lapse series capturing anther closure and color change were created. Video S1 was made

| Floral observations and natural rainfall
Observations of anthesis-focused on androecial maturation, anther dehiscence, and pollen shedding-were recorded for 542 flowers total over the course of our 60-day study. Of these, records for 364 flowers (67%) were "complete" (as defined in Methods) and, thus,

| Simulated rainfall experiment
Over the 30 min of simulated rainfall, a rain gauge in the water- Similarly, after exposure to the simulated rainfall treatment, 57% of flowers had lighter colored anthers and 43% of flowers had anthers with no change in pigmentation (including some that were beige before the simulated rainfall). In the control group, 11% of flowers had lighter colored anthers, 6% of flowers had more pigmented anthers, and 83% of flowers had anthers with no change in pigmentation. The difference between the experimental and control groups was significant (X 2 = 5.31, df = 1, p = .021; data not shown).

| Time-lapse photography
We took time-lapse series to document the changes in R. hermaphrodita anthers following water exposure. In our videos, water droplets caused anther walls to swell rapidly and approximately double in size as they bent inwards towards their line of dehiscence, enveloped pollen, and finally closed completely as they reached terminal size (Videos S1 and S2). This occurred after 5-10 min of intermittent water exposure. Anther depigmentation occurred more slowly and-in contrast to anther position-continued after anthers reached their terminal size. At the end of Video S1, which was shot over 22:30 min, anthers were lighter in color, but still tinted purple.

F I G U R E 3 Changes in anther position
At the end of Video S2, which was shot over 67:30 min, anthers were fully translucent.

| Floral observations and natural rainfall
For half of the rain events (5 of 10), we observed associated transitions in anther color and position across >88% of flowers (Figure 3, blue arrows). Average rainfall was more than four times higher for rain events with associated anther change than for rain events without associated changes. Given this, it seems likely that larger amounts of rain produce a stronger, more consistent response in anthers. With the weather station roughly 16 km away from our study site, it is possible that a precipitation event, especially a small one, could pass over one location and not the other or that rainfall intensity at the two locations could vary. We hypothesize this is why we did not see floral change associated with every rain event recorded at the weather station. While this is a limitation for our natural rainfall data, the prevailing trends in these data-that R. hermaphrodita anthers close and lose color after rain-are further substantiated by the results of our simulated rainfall experiment and time-lapse photography.
From August 11 onward, we noted whether the anthers we observed were covered with water, giving us a directly observed indicator for rainfall at the study site. There were three events where most of the anthers were enveloped in a film of water. These events correspond exactly with the three broad transitions in anther color and position observed during that period. This close association lends further support to the idea that differences in weather at the study site versus the weather station explain the five events without correlated anther closure and color change.

| Anther closure
When anthers open, the final outward bending of the anther walls is often facilitated by dehydration (Keijzer, 1987;Keijzer et al., 1996;Wilson et al., 2011). This dehydration can be the result of evaporation, reabsorption, or both (Timerman & Barrett, 2021), and, in some cases, may be related to the sudden drop in relative humidity that anthers experience upon floral anthesis (Keijzer et al., 1996). This may explain why we observed R. hermaphrodita F I G U R E 4 Simulated rainfall induces anther closure and color change in Ripariosida hermaphrodita. (a) Anther position and (b) color before and after simulated rainfall treatment. The differences between the experimental (14 flowers) and control (18 flowers) groups for both anther position and anther color were shown to be statistically significant.
Asterisks indicate a p-value of <.01.
anthers opening immediately after buds opened and petals unfurled. In Laurus nobilis, Lilium philadelphicum, and Butomus umbellatus, rainfall has been suggested to lead to the rehydration of various anther tissues that then cause anthers to reclose (Edwards & Jordan, 1992;Li et al., 2012;Pacini et al., 2014). These tissues include valve cells (Pacini et al., 2014), the epidermis (Edwards & Jordan, 1992;Li et al., 2012), and the endothecium (Edwards & Jordan, 1992). In at least one case, this process occurs passively (Pacini et al., 2014). It is likely a similar mechanism that underlies anther closure in R. hermaphrodita.
Some plants have been known to reopen closed anthers, including Mirabilis nyctaginea (Cruden, 1973), Butomus umbellatus (Li et al., 2012), and Paris polyphylla var. yunnanensis, the last of which, in addition to closing anthers in response to rain, closes its anthers every night and reopens them in the morning (Wang et al., 2009). In Lilium philadelphicum, which has anthers that close during rainfall, it is thought that pollen viability extends through floral senescence (Edwards & Jordan, 1992) and for Butomus umbellatus, it has been shown that anther closure during rain extends pollen viability (Li et al., 2012). In contrast, R. hermaphrodita anthers do not reopen after rainfall and thus their closure can provide little male fitness advantage to the species. This is further supported by the relatively short flowering period of any In the past century, anther closure after rainfall has been little studied. To our knowledge, since Kerner von Marilaun's compilation (1895), only four additional species have been noted as displaying rainfall-induced anther closure (Cruden, 1973;Edwards & Jordan, 1992;Li et al., 2012;Wang et al., 2009). In this report, we add a fifth. By and large, modern methods have not been used to compare anther closure across species or investigate the extent to which evolutionary pressures are behind this phenomenon, leaving a significant gap in the literature. is the potential that the effects of anther color change could be complicated by multiple pollinators responding to different floral cues, for example, the attraction of flies to beige/white floral parts versus bees to purple/blue floral parts (Richards, 1997).

| Anther color change
Therefore, pollinator preferences could enact different, and perhaps contradictory, selective forces on the flowers in this species.
Furthermore, for a full picture of the potential push and pull of reproductive evolutionary pressures on R. hermaphrodita, other factors must also be considered, such as the timing and volume of nectar production (i.e., another pollinator reward besides pollen), as well as the timing and duration of stigma receptivity (i.e., female fitness), which appears to occur towards the end of pollen release (Humphreys, personal observations).
There are three main classes of pigments associated with floral color change: betalains, carotenoids, and anthocyanins (Weiss & Lamont, 1997). In plants, betalains are exclusively found in the order Caryophyllales (Grotewold, 2006), so this class is not relevant for R. hermaphrodita. While carotenoids create yellow and orange floral hues, anthocyanins create orange, red, purple, and blue colors (Grotewold, 2006). Anthocyanins are water soluble and changes in pH affect anthocyanin color and create not only shifts between red and blue, but also colorless compounds (Enaru et al., 2021;Grotewold, 2006;Weiss & Lamont, 1997). Given all these factors, we hypothesize that the transition from purple to beige observed in R. hermaphrodita anthers is most likely the result of a rainfall-induced pH change or a loss of anthocyanins. Further investigation is needed to determine the exact mechanism of this observed color change.